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. 2018 Feb 28;28(9):1704598.
doi: 10.1002/adfm.201704598. Epub 2018 Jan 5.

Nanoparticle-based fluoroionophore for analysis of potassium ion dynamics in 3D tissue models and in vivo

Bernhard J Mueller  1 , Alexander V Zhdanov  2 , Sergey M Borisov  1 , Tara Foley  3 , Irina A Okkelman  2 , Vassiliy Tsytsarev  4 , Qinggong Tang  5 , Reha S Erzurumlu  4 , Yu Chen  5 , Haijiang Zhang  6 , Claudio Toncelli  6 , Ingo Klimant  1 , Dmitri B Papkovsky  2 , Ruslan I Dmitriev  2
Affiliations

Nanoparticle-based fluoroionophore for analysis of potassium ion dynamics in 3D tissue models and in vivo

Bernhard J Mueller et al. Adv Funct Mater. .

Abstract

The imaging of real-time fluxes of K+ ions in live cell with high dynamic range (5-150 mM) is of paramount importance for neuroscience and physiology of the gastrointestinal tract, kidney and other tissues. In particular, the research on high-performance deep-red fluorescent nanoparticle-based biosensors is highly anticipated. We found that BODIPY-based FI3 K+-sensitive fluoroionophore encapsulated in cationic polymer RL100 nanoparticles displays unusually strong efficiency in staining of broad spectrum of cell models, such as primary neurons and intestinal organoids. Using comparison of brightness, photostability and fluorescence lifetime imaging microscopy (FLIM) we confirmed that FI3 nanoparticles display distinctively superior intracellular staining compared to the free dye. We evaluated FI3 nanoparticles in real-time live cell imaging and found that it is highly useful for monitoring intra- and extracellular K+ dynamics in cultured neurons. Proof-of-concept in vivo brain imaging confirmed applicability of the biosensor for visualization of epileptic seizures. Collectively, this data makes fluoroionophore FI3 a versatile cross-platform fluorescent biosensor, broadly compatible with diverse experimental models and that crown ether-based polymer nanoparticles can provide a new venue for design of efficient fluorescent probes.

Keywords: Bionanotechnology; Core/Shell Nanoparticles; Live cell imaging; Medical Applications; Sensors/Biosensors.

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Conflict of interest statement

Competing financial interests None declared.

Figures

Figure 1
Figure 1
Scheme and spectral properties of K+-sensitive FI3 dye encapsulated in RL100 nanoparticles. Spectra were measured in 20 mM Tris-HCl, pH 7.4 with concentration of nanoparticles adjusted to A630 =0.1.
Figure 2
Figure 2. FI3 nanoparticles provide efficient staining of various 2D and 3D tissue models
A: adherent cell models of neuronal and non-neuronal origin. Live cells were incubated with FI3 nanoparticles (10 μg/ml, 16 h), washed and counter-stained with Calcein Green AM (1 μM, 0.5 h) and imaged. B: staining of rat primary neurospheres and mouse intestinal organoids. Live neurospheres and organoids were incubated with FI3 nanoparticles (10 μg/ml) for 16 h and 3 h, respectively, counter-stained with Calcein Green or Hoechst 33342 and imaged. C: staining of tumor spheroids from HCT116 cells. Formed tumor spheroids were incubated with FI3 nanoparticles (3 h) and counter-stained with Calcein Green. The image represents cross-section at depth 50 μm, as indicated. Right graph shows distribution of fluorescence intensity in the center of spheroids at depths 0-50 μm. BG indicates background intensity. Scale bar is in μm.
Figure 3
Figure 3. FI3 dye remains encapsulated in the nanoparticles after cell internalization
A-B: comparison of staining efficiency of live primary neurons with FI3 as free dye and encapsulated in nanoparticles (NP). The dye concentration of 0.1 μM equals to 10 μg/ml of nanoparticles. Cells were incubated with dye and dye/NP at indicated concentrations (16 h), washed and imaged. A: Comparison of fluorescence intensity in cells for the free dye and NP at different concentrations. B: Comparison of photostability between free dye, NP and Calcein Green. N=8. C-F: The differences in fluorescence lifetimes observed in cells stained with free dye and NP, measured by FLIM. C: characteristic images of mixed culture of astro-glial cells and neurons stained with FI3. Intensity (red fluorescence), co-staining with Calcein Green and FLIM images are shown. D: typical fluorescence lifetime decay curves (dots) and the respective fits (lines), shown in logarithmic scale. E, F: distribution histograms for free dye and NP, observed for primary neurons and HCT116 cells. N = 3. Scale bar is in μm.
Figure 4
Figure 4. Mechanism of cell entry for FI3 nanoparticles
A: Staining kinetics for FI3 (0.1 μM free dye or 10 μg/ml RL100 nanoparticles) in live rat primary neural cells. Cells were incubated with FI3 for indicated time intervals, washed and immediately imaged. B-D: Staining of primary neural cells with FI3 nanoparticles (10 μg/ml, 3 h). B: Effect of temperature on staining efficiency. C: Effects of various endocytosis inhibitors on cell staining with FI3. Cells were pre-treated with inhibitors (50 μM EIPA, 10 μg/ml CPZ and 5 mg/ml MβCD) for 30 min, followed by staining procedure. MβCD-treated cells displayed round morphology, indicated with green arrows. N= 17. D: Effect of ATP depletion on cell staining efficiency. For ATP depletion, cells were pre-incubated in no-glucose medium and treated with oligomycin (10 μM), followed by staining procedure. N=20. E: Cell staining efficacy for K-FI3 and Na-FI3. Cells were incubated with nanoparticles (10 μg/ml, 16 h) washed, counter-stained with Calcein Green and imaged. Scale bar is in μm.
Figure 5
Figure 5. Monitoring of extra- and intracellular K+ in neural cells with FI3 nanoparticles
The live rat primary neural cells were stained with nanoparticles (10 μg/ml, 16 h), counter-stained with Calcein Green and treated with KCl and valinomycin (Val). A: Representative transmission light (DIC) and superimposed confocal fluorescence images of FI3 and Calcein before and after treatment with KCl (40 mM) and valinomycin (Val, 0.5 μM). B-C: Line profile analysis of the changes in FI3 and Calcein fluorescence signals, induced by treatments in (A). The areas of intra- and extracellular FI3 pools are highlighted in (B) in grey and white, respectively. D. Results of line profile analysis of the changes in intracellular FI3 signal, induced by the sequential addition of Val and KCl. Images (A) represent stacks of 2 confocal planes taken with 0.5 μm step. Error bars indicate SEM. Asterisks indicate significant difference (p < 0.01, U test). N = 4 (D). Scale bar is in μm.
Figure 6
Figure 6. Application of FI3 nanoparticles to ex vivo and in vivo brain imaging
A-B: staining efficiency and distribution of FI3 (10 μg/ml, 6 h) in live organotypic rat brain slices. Cortex region is shown. Staining with FI3 (red) and cholera toxin-Alexa Fluor 488 conjugate (green) is shown. B: 3D reconstruction (the views of XY and Z projections) of FI3 and CTX localization in the tissue, and corresponding line profile analysis. Images represent stacks of 21 (A) and 16 (B) confocal planes taken with 2 μm step. C-D: In vivo imaging of stained mouse brain. C: Pseudocolor images of the cortex before and after stimulus onset. Time (ms) after beginning of the trial is indicated by numbers. D: Time-course of recorded response at the locations (depicted in C) having different distances from the electrode.
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